1. Field of the Invention
The invention relates generally to fluorescent proteins and more particularly to compositions and methods for measuring the response of a sensor polypeptide to an environmental (e.g., biological, chemical, electrical or physiological) parameter.
2. Background Information
Fluorescent Ca2+ indicators such as fara-2, indo-1, fluo-3, and Calcium-Green have been the mainstay of intracellular Ca2+ measurement and imaging (see, for example, U.S. Pat. No. 4,603,209 and U.S. Pat. No. 5,049,673). These relatively low molecular weight indicators can suffer from many technical problems relating to ester loading, leakage of the dyes from the cell, compartmentalization in organelles, and perturbation of the indicators by cellular constituents. Although the Ca2+-indicating photoprotein aequorin is targetable, the photoresponse to Ca2+ is low because it is chemiluminescent. Moreover, aequorins need to incorporate exogenous coelenterazine.
Many effects of Ca2+ in cells are mediated by Ca2+ binding to calmodulin (CaM), which causes CaM to bind and activate target proteins or peptide sequences. Based on the NMR structure of CaM bound to the 26 residue M13 Ca2+-binding peptide of myosin light-chain kinase, Porumb et al. fused the C-terminus of CaM via a Gly-Gly spacer to M13. Ca2+ binding switched the resulting hybrid protein (CaM-M13) from a dumbbell-like extended form to a compact globular form similar to the CaM-M13 intermolecular complex (see, Porumb et al., Prot. Engineering 7:109–115 (1994)).
Measurement of a binding member concentration in vitro or in vivo by non-invasive techniques can help elucidate the physiological function of the binding member. This can also aid in identifying changes that occur in a cell or organism in response to physiological stimuli. For example, cyclic AMP can be detected by fluorescence resonance energy transfer between separately labeled proteins that associate with each other but are not covalently attached to each other (see, U.S. Pat. No. 5,439,797).
The Aequorea victoria Green Fluorescent Protein (GFP) is useful as a marker for gene expression, as a fluorescent tag to aid in visualizing protein trafficking, and as a component of indicator systems that allow fluorescent sensing of small molecules and pH. Currently, the use of GFPs as a biosensor is limited to those systems that use GFP fusion proteins as partners for fluorescence resonance energy transfer (FRET) or those that use the subcellular redistribution of GFP fusion proteins as indicators of substrate concentration or the measurement of pH.
Currently, fluorescent molecules designed to measure interactions of proteins rely on cameleon molecules of tandem GFP constructs. In these constructs, conformational changes occur and alter the FRET between the GFPs such that a ratiometric color change is noted. Such cameleon or FRET-sensitive constructs are large molecules, in which protein conformation influences FRET efficiency of two GFPs of different colors. Although insertions into Green Fluorescent Protein have been attempted (see Abedi et al., Nucleic Acids Research, 26(2):623–630 (1998)), such insertions have been made to optimize the presentation of short peptide libraries and not to present binding molecules or sensor polypeptides. Additionally, such insertions have been only short insertions of about six amino acids in length. Until now, however, it has not been possible to make a single GFP molecule fluorescence-sensitive to a substrate other than hydrogen ions. Thus, there currently is a desire for smaller constructs useful in measuring interactions of molecules in vitro and in vivo.